Follicle stimulating hormone (FSH) is known to be useful in the treatment of infertility. Preparations containing this hormone have been employed to assist in effecting pregnancy using both in-vivo and in-vitro techniques. Human FSH has been isolated from human pituitary glands and from post-menopausal urine. More recently, it has been produced using recombinant DNA techniques.
The first commercially available product comprising human FSH contained HMG (e.g., Pergonal.RTM. Serono), i.e., human menopausal gonadotropin extracted from post-menopausal urine which is a mixture of FSH, Luteinizing Hormone (LH) and other urinary proteins. Meanwhile, several attempts were made to obtain pure FSH preparations, both for scientific and therapeutic purposes. The product Metrodin.RTM. (Serono), currently available in commerce, is a preparation of urinary FSH containing other urinary proteins, but minimum quantities of LH and is used for the treatment of infertility. Use of this product is particularly advantageous when administration of exogenous LH together with FSH is undesirable, e.g., in the polycystic ovary syndrome (PCOS).
So far, administration of FSH for therapeutic purposes has been carried out, successfully, exclusively by intramuscular injection. Since intramuscular injections are generally performed by the physician or by the medical professional staff, the patient is expected to visit a surgery or a hospital regularly in order to receive the treatment. This creates a considerable discomfort. Moreover, the time taken up by this type of application often leads to unsatisfactory compliance by the patient as the treatment normally extends over several weeks or months.
Administration by subcutaneous injection would render possible the self-administration by the patient and consequently improve patient's cooperation and compliance.
The subcutaneous administration of Human Menopausal Gonadotropin (HMG) has already been described (Nakamura Y. et al., Fertility and Sterility, 46(1):46-54, 1986) in connection with the treatment of female infertility by pulsatile administration of HMG via the subcutaneous peristaltic pump. The subcutaneous administration may suffer the drawback of the appearance of local allergies due to the presence of impurities in the product used and, consequently, result in the suspension of the treatment.
P. Roos ("Human Follicle Stimulating Hormone", Acta Endocrinologica Supplementum 131, 1968) described and characterized highly purified preparations of pituitary and urinary FSH obtained from frozen pituitaries and from post-menopausal urinary concentrate, respectively. Biological potencies as high as about 14,000 I.U. of FSH activity per mg for pituitary FSH and 780 I.U. of FSH activity per mg for urinary FSH were obtained. The content of LH contamination in the most active pituitary and urinary preparations was estimated to correspond to approximately 0.1 percent by weight. The purification procedures involved one or more of such techniques as chromatography on DEAE-Cellulose, gel-filtration on Sephadex G-100, hydroxylapatite chromatography, polyacrylamide gel electrophoresis, and the like.
One of the best purified urinary FSH preparations was described by Donini et al. ("Purification and partial Chemico-physical characterization of FSH from Menopausal Urine", Gonadotrophins and Ovarian Development (Proceedings of two workshop meetings), E and S Livingstone, Edinburgh and London, 1970) and had a biological potency of 1255.6 I.U. FSH per mg with an LH contamination as low as 3.2 I.U. LH per mg.. In this case, the starting material was a Human Menopausal Gonadotropin (HMG) preparation (Pergonal.RTM.) which, as stated above, is a mixture of FSH and LH hormones and other urinary proteins. This result was achieved by batchwise purification of the starting HMG on DEAE-Cellulose followed by chromatography on a DEAE-Cellulose column, gel filtration on Sephadex G-100 and a final step of preparative polyacrylamide gel electrophoresis.
Even higher biological potencies were achieved by H. Van Hell et al. ("Purification and some properties of human urinary FSH and and LH", Gonadotropins (Proceedings of Int.l Symposium on Gonadotropins, 1971), Wiley-Interscience, New York) by adding immunochromatography and gel-filtration steps to a conventional chromatographic purification procedure. Immunochromatography was performed using anti-HCG antibodies coupled to Sepharose for the specific purpose of removing the LH activity from partially purified FSH fractions. The best purified FSH fraction contained 4720 I.U. FSH per mg and the LH contamination was as low as 15 I.U. LH per mg as assayed by RIA.
The immunochromatographic approach had already been described by Donini et al. ("Purification and Separation of FSH and LH from HMG", Acta Endocrinologica 52, pages 186-198, 1966) who obtained as early as 1966 an FSH preparation which had a potency of 329.7 I.U. FSH per mg and biologically undetectable amounts of LH contamination. In another experiment, a fraction assaying 148.3 I.U. FSH per mg and 2.4 I.U. LH per mg was obtained. Since no RIA was performed on the 329.7 I.U. FSH per mg preparation, it can be assumed by analogy with the results of H. Van Hell (c.f. supra) that traces of LH would have been found if assayed by RIA.
The physiological relevance of even minimal amounts of LH contamination was shown by Donini et al. in a paper ("A new approach to the Biological Determination of the Luteinizing Hormone", Acta Endocrinologica, 58, pages 463-472, 1968) where a new bio-assay for LH determination was proposed which consisted in injecting intact immature mice with a constant dose of a biologically pure FSH preparation plus increasing amounts of LH and then measuring the increase in the uterine weight as a response proportional to the LH activity. By this method, as little as 0.068 I.U. of LH were shown to be capable of increasing the uterine weight when injected together with 4.44 I.U. of FSH.
It thus clearly follows that an absolutely pure FSH preparation is desirable when FSH activity in the complete absence of LH activity is requested in therapy.
As mentioned above, the currently marketed product Metrodin.RTM. (Serono) is a purified FSH preparation which is obtained by a method substantially identical to that described by Donini et al. in an already mentioned paper (Acta Endocrinologica 52, pages 186-198, 1966). The quality control specifications, in agreement with the declared biological purity of the said preparation, provide for not more than 0.7 I.U. LH per 75 I.U. of FSH, i.e., approximately the sensitivity limit of the biological assay. In certain therapeutic applications, however, the absolute absence of LH is desirable. Furthermore, in Metrodin.RTM., human FSH is accompanied by substantial amounts of other urinary proteins, i.e., it is not a chemically pure FSH preparation.
U.K. patent application 2,173,803 A provides still another approach to the purification of pituitary glycoprotein hormones, among them FSH. This approach consists of first forming a complex of the hormone with immobilized monoclonal antibodies and then eluting the hormone with an acidic aqueous buffer having a pH from 3 to 4. As far as FSH is concerned, the obtained highly purified hormone is still contaminated with 0.1% by weight of LH and 0.5% by weight of TSH.
Scott C. Chappel et al. describe in a review article (Endocrine Reviews 4(2), 179-211, 1983) the microheterogeneity of FSH and the physiological significance of the carbohydrate moieties accompanying the FSH molecule. The hypothesis can be formulated that some modifications occur during the metabolic pathway up to the final urinary secretion which may account for the chemico-physical differentiation demonstrated in the experiments carried out by Applicant between the highly purified urinary FSH preparation (hpuFSH) according to this invention, and the highly purified pituitary FSH preparation used for comparison purposes.
Co- or post-translationally modifications appear to be the basis for microheterogenous diversity of FSH, a glycoprotein consisting of two dissimilar, glycosylated, non covalently linked polypeptide chains known as alpha and beta-subunit. The beta-subunit endows the molecule with its biological specifity.
Incidentally, the same article attempts to explain the substantial difference in the values of biological activity per mg between highly purified pituitary and urinary FSH preparations. A reasonable hypothesis can again be based on the relevance of the carbohydrate moieties and, more specifically, the role of the terminal sialic acid residues.
It should be noted that, while both pituitary FSH and urinary FSH are termed "FSH", no conclusive evidence has yet been found which verifies whether the molecules isolated from the two sources are the same or even chemically equivalent.